Isothermal Amplification of Nucleic Acid, and Library Preparation and Clone Generation in Sequencing

Abstract

An amplification system that provides methods and reaction components that allow for isothermal amplification for detection of target nucleic acid 24 ; allow non-enzymatic amplification for detection of target nucleic acid 24 ; can be used to identify amplicons without having to create separate individual probes for each target nucleic acid 24 , and can be used to improve sequencing processes.

What is claimed is: 
     
         1 . A process for detecting the presence of a target nucleic acid by amplifying at least one target nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids, the process comprising:
 bringing a nucleic acid or a mixture of nucleic acids into contact with a probe, said probe including:
 a first segment having a sequence that is complementary to a target nucleic acid, and 
 a second segment having a sequence complementary to the sequence of a primer wherein the sequence of the primer is adapted to conform into a conformation having a free energy with more favorable thermodynamics than a corresponding B-DNA duplex; and 
   bringing said nucleic acid or mixture of nucleic acids into contact with said primer, wherein said primer will bind said second segment of said probe and will subsequently conform into said conformation having a free energy with more favorable thermodynamics than a corresponding B-DNA duplex during an extension step of a polymerase chain reaction under isothermal conditions.   
     
     
         2 . The process of  claim 1 , wherein the conformation having a free energy with more favorable thermodynamics than a corresponding B-DNA duplex is a quadruplex. 
     
     
         3 . The process of  claim 1 , the primer having a G content of an amount to conforms into a quadruplex structure during an extension step of a polymerase chain reaction. 
     
     
         4 . The process of  claim 3 , wherein the G content is equal to or greater than 70%. 
     
     
         5 . The process of  claim 1 , wherein at least a portion of the primer has a sequence based on (G 3+ N 1-7 G 3+ N 1-7 G 3+ N 1-7 G 3+ ). 
     
     
         6 . The process of  claim 1 , wherein at least a portion of the primer has a sequence based on (GGGT) 4  [SEQ. ID. NO. 5]. 
     
     
         7 . The process of  claim 1 , the primer further including in its sequence at least one label chosen from 2Ap, 3MI, 6MI, and 6AMP. 
     
     
         8 . The process of  claim 1 , wherein the probe further includes a third segment having a sequence adapted to conform into a conformation having a free energy with more favorable thermodynamics than a corresponding B-DNA duplex, wherein said primer binds said second segment of said probe and, during an extension step of a polymerase chain reaction, will unfold said third segment of said probe. 
     
     
         9 . The process of  claim 8 , wherein said third segment of said probe and said extended primer will both subsequently conform into said conformation having a free energy with more favorable thermodynamics than a corresponding B-DNA duplex during an extension step of a polymerase chain reaction under isothermal conditions. 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . The process of  claim 9 , wherein at least a portion of the third segment of the probe and the primer each has a sequence based on (G 3+ N 1-7 G 3+ N 1-7 G 3+ N 1-7 G 3+ ). 
     
     
         14 . The process of  claim 9 , wherein at least a portion of the third segment of the probe and the primer each has a sequence based on (GGGT) 4  [SEQ. ID. NO. 5]. 
     
     
         15 . The process of  claim 9 , the third segment of the probe and the primer each further include in its sequence at least one label chosen from 2Ap, 3MI, 6MI, and 6AMP. 
     
     
         16 . A The process of  claim 1 , further comprising:
 bringing the nucleic acid or a mixture of nucleic acids into contact with a second probe, said second probe including a first segment having a sequence that is complementary to a second target nucleic acid, and a second segment having a sequence complementary to the sequence of a second primer, wherein the sequence of the second primer is adapted to conform into a conformation having a free energy with more favorable thermodynamics than a corresponding B-DNA duplex; and   bringing said nucleic acid or mixture of nucleic acids into contact with said second primer, wherein said second primer will bind said second segment of said second probe and will subsequently conform into said conformation having a free energy with more favorable thermodynamics than a corresponding B-DNA duplex during an extension step of a polymerase chain reaction under isothermal conditions.   
     
     
         17 . (canceled) 
     
     
         18 . The process of  claim 16 , the second segments of the first and second probes having a G content of an amount to conforms into a quadruplex structure during an extension step of a polymerase chain reaction. 
     
     
         19 . The process of  claim 18 , wherein the G content is equal to or greater than 70%. 
     
     
         20 . The process of  claim 16 , wherein at least a portion of the second segments of the first and second probes has a sequence based on (G 3+ N 1-7 G 3+ N 1-7 G 3+ N 1-7 G 3+ ). 
     
     
         21 . The process of  claim 16 , wherein at least a portion of the second segments of the first and second probes has a sequence based on (GGGT) 4  [SEQ. ID. NO. 5]. 
     
     
         22 . The process of  claim 16 , the second segments of the first and second probes further including in its sequence at least one label chosen from 2Ap, 3MI, 6MI, and 6AMP. 
     
     
         23 . A process for preparing a library of a target nucleic acid for sequencing by amplifying the target nucleic acid sequence, the process comprising:
 bringing a nucleic acid into contact with a first primer, said first primer immobilized to a surface, and said nucleic acid including first, second, third, and fourth segments:
 the first segment having a sequence that is complementary to the first primer immobilized to the solid support;
 the second segment having a sequence of the target nucleic acid, or complementary to the sequence of the target nucleic acid; 
 
 the third segment having a sequence complementary to a sequencing primer; and 
 the fourth segment having a sequence adapted to conform into a conformation having a free energy with more favorable thermodynamics than a corresponding B-DNA duplex; 
   wherein binding between the first segment and the first primer results in the generation of a complementary strand to the nucleic acid;   bringing said nucleic acid and said complementary strand into contact with a second primer, wherein said second primer is adapted to conform into a conformation having a free energy with more favorable thermodynamics than a corresponding B-DNA duplex during an extension step of a reaction under isothermal conditions; and   bringing said nucleic acid and said complementary strand into contact with said sequencing primer.   
     
     
         24 . (canceled) 
     
     
         25 . (canceled) 
     
     
         26 . A process for preparing a library of a target nucleic acid for sequencing by amplifying the target nucleic acid sequence, the process comprising:
 bringing a nucleic acid into contact with a first primer, said first primer immobilized to a surface, wherein said first primer is adapted to conform into a conformation having a free energy with more favorable thermodynamics than a corresponding B-DNA duplex during an extension step of a reaction under isothermal conditions, and wherein said nucleic acid includes first, second, third, and fourth segments:
 the first segment having a sequence that is complementary to the first primer immobilized to the solid support;
 the second segment having a sequence of the target nucleic acid, or complementary to the sequence of the target nucleic acid; 
 
 the third segment having a sequence complementary to a sequencing primer; and 
 the fourth segment having a sequence complementary to a second primer; 
   wherein binding between the first segment and the first primer results in the generation of a complementary strand to the nucleic acid;   bringing said nucleic acid and said complementary strand into contact with said second primer; and   bringing said nucleic acid and said complementary strand into contact with said sequencing primer.   
     
     
         27 . (canceled) 
     
     
         28 . (canceled)